Bacterial-mediated transformation via, for example, Agrobacterium or Rhizobium, entails the transfer and integration of a polynucleotide from a bacterial plasmid into the genome of a eukaryotic organism. The region of DNA within the bacterial plasmid that is designated for such manipulation is called the transfer DNA (“T-DNA”).
A T-DNA region is delimited by left and right “border” sequences, which are each about twenty-five nucleotides in length and oriented as imperfect direct repeats of the other. T-DNA transfer is initiated by an initial single stranded nick at the so-called right border site and is terminated by a subsequent secondary nick at the left border site. It is the resultant single-stranded linear DNA molecule that is transported, by the activity of other proteins, into the plant cell and ultimately integrated into the plant genome.
After initial cleavage at the right border, virD2 covalently binds to the 5′-side, and the DNA unwinds towards the left border where a second cleavage reaction occurs. The released single stranded DNA, traditionally referred to as the “T-strand,” is coated with virE2 and processed for transfer via type IV type secretion (Lessl and Lanka, (1994) Cell 77: 321-324, 1994; Zupan and Zambryski, Plant Physiol 107: 1041-1047, 1997).
Since border sequences alone do not support a highly effective DNA transfer, extended border regions, generally comprising about 200 or more base pairs of Agrobacterium tumor-inducing (Ti) plasmid DNA, are used to transform plant cells. Two non-border sequences that are located within these extended border regions have been shown to promote DNA transfer, namely the ‘overdrive’ domain of pTi15955 (van Haaren et al., Nucleic Acids Res. 15: 8983-8997, 1987) and a DNA region containing at least five repeats of the ‘enhancer’ domain of pRiA4 (Hansen et al., Plant Mol. Biol., 20:113-122, 1992).
One issue associated with the use of conventional Agrobacterium border regions is the infidelity of DNA transfer. For instance, primary cleavage reactions at the right border are often not followed by secondary cleavage reactions at the left border. This “border skipping” leads to the transfer of T-DNAs that are still connected to the rest of the plasmid. Such plasmid backbone transfer is undesirable because these sequences typically comprise antibiotic resistance genes. Plasmid backbone transfer can also be a consequence of inadvertent right border activity at the left border.
A second issue concerns the use of conventional and poorly characterized Agrobacterium border regions, which permit only very little optimization of transfer frequencies. This leads to poor transformation rates, and high input costs for the production of large numbers of transformed plants.
Furthermore, the presence of foreign T-DNA sequences in food crops is often perceived as undesirable, and the application of genetic engineering has therefore been limited to a small number of crops that are destined for feed, oil, fibers, and processed ingredients. Public concerns were addressed through development of an all-native approach to making genetically engineered plants, as disclosed by Rommens et al. in WO2003/069980, US-2003-0221213, US-2004-0107455, and WO2005/004585, which are all incorporated herein by reference. Rommens et al. teach the identification and isolation of genetic elements from plants that can be used for bacterium-mediated plant transformation. Thus, Rommens teaches that a plant-derived transfer-DNA (“P-DNA”), for instance, can be isolated from a plant genome and used in place of an Agrobacterium T-DNA to genetically engineer plants.
The concept of P-DNA mediated transformation has previously been demonstrated in potato. A 400-base pair potato P-DNA delineated by regions that share sequence identity with the left border of nopaline strains and the right border of octopine strains was effectively transferred from Agrobacterium to plant cells (Rommens et al., Plant Physiol 135: 421-431, 2004).
The potato P-DNA was subsequently used to introduce a silencing construct for a tuber-specific polyphenol oxidase (PPO) gene into potato. Resulting intragenic plants displayed tolerance against black spot bruise sensitivity in impacted tubers.
The present invention provides new plant-specific DNA elements that replace bacterial borders, and are particularly useful for all-native DNA transformation methods.
The present invention also reveals the organization of the extended regions that are involved in the initiation of DNA transfer by mediating primary DNA cleavage, and describes the sequence requirements and spacing of genetic elements that support high activity of the described elements. Furthermore, the invention shows how manipulations of regions that surround enzyme cleavage sites can enhance the fidelity of DNA transfer.